CRISPR/Cas9-mediated viral interference in plants
Item Type Article
Authors Ali, Zahir;Abulfaraj, Aala A.;Idris, Ali;Ali, Shawkat;Tashkandi, Manal;Mahfouz, Magdy M.
Citation CRISPR/Cas9-mediated viral interference in plants 2015, 16 (1) Genome Biology
Eprint version Publisher's Version/PDF
DOI 10.1186/s13059-015-0799-6
Publisher Springer Nature
Journal Genome Biology
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creativecommons.org/licenses/by/4.0/), which permits
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Download date 2023-10-31 16:35:21
Link to Item http://hdl.handle.net/10754/582543
SUPPLEMENTARY INFORMATION: ADDITIONAL FILE 1 CRISPR/Cas9-mediated viral interference in plants
Zahir Ali1, Aala Abulfaraj1, Ali Idris1, Shakila Ali1, Manal Tashkandi1, and Magdy M.
Mahfouz1, 2
1 Laboratory for Genome Engineering, Center for Desert Agriculture & Division of
Biological Sciences, 4700 King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia
Supplementary Tables
Supplementary Table 1. Primers used in this study.
Supplementary Table 2. Summary of different sgRNA used for targeting of TYLCV genome.
Supplementary Table 1. Primers used in this study.
primers name sequence (5- ---- 3-) Usage
TYLCV2.3-IR-T-F AATTGGGAAAGTGCTTCCTCT Simi-q PCR, to amplify TYLCV IR flanking region, to make probe for
southern, detection TYLCV by PCR TYLCV2.3-IR-T-R ATAGTCACGGGCCCTTACAACA
TYLCV-IR-T1 CGAGTCTAGAGGCCATCCGTATA ATATTACGTTTTAGAGCTAGAAA TAGCAAG
To clone TYLCV IR- gRNA SPDK-gRNA-R acatGCCCGGgAAAAAAAGCACCG
ACTCGG
To clone all gRNA NB-ACTIN1-RT-F TGAAGATCCTCACAGAGCGTGG RT-PCR normalization
control NB-ACTIN1QRT-LIU-R TTGTATGTGGTCTCGTGGATTC
TYLCV-CP-T1 CGAGTCTAGAGCTTCGGCGAACC TTCGAGACGTTTTAGAGCTAGAA ATAGCAAG
To clone TYLCV CP- gRNA
TYLCV-RCRII-T CGAGTCTAGAGTGGATGAGCACA TGCAAGTGGTTTTAGAGCTAGAA ATAGCAAG
To clone TYLCV RCRII-gRNA TRV1-RELICASE-RT-F CTACTGGGAGAGCAGCAACC For detection of TRV-
RNA1 systemic movement TRV1-REPLICASE-RT-R CTGAGCGCAAAAGTACACCA
TRV2-CP-RT-F TTGGGTGGAATCAGTTTCGT For detection of TRV- RNA2 systemic
movement TRV2-CP-RT-R TCTTCCAAAGTCGAGCCAGT
WOR-IR-T-F GGCTTTAATTTGAAATGATGGTG For PCR flanking Worland IR target WOR-IR-T-R AAAAATTCGTACCTGATTGCAG
WOR-IR-T1 CGAGTCTAGAGCCATCCGCAATAATATTACG TTTTAGAGCTAGAAATAGCAAG
To clone Worland IR- gRNA
WOR-L1-T1/2-F TGCTTCAGCTGCATTACCTG For PCR flanking Worland RCRII target WOR-L1-T1/2-R ATGGCCCCTGGAGGTATATAAG
WOR-LI-RCRII-T GAGTCTAGAGCTTTGAATTGGATGAGGGCG GTTTTAGAGCTAGAAATAGCAAG
To clone Worland RCRII- gRNA
TYLCV 2.3-CP-T1/2-F TTCTTCACGGTTGCGGTACT For PCR flanking TYLCV CP target TYLCV2.3-CP-T1/2-R GAGCTTTGGACCCTGAATTG
TYLCV2.3-REP-T1/2-F GAGCTTTGGACCCTGAATTG For PCR flanking TYLCV RCRIII target TYLCV2.3-REP-T1/2-R TTGGAGCGTGATGATTTTGA
MeMV-IR-T-F TGTGCAGAGCTTTGATTTGG For PCR flanking MeMV IR target MeMV-IR-T-R AAATTGGCGTTGCGACTAAC
Supplementary Table 2. Summary of different sgRNA used for targeting of TYLCV genome. All the observations were performed with NB- Cas9OE plants. Data were collected at 28 dpi. Three biologically independent experiments were performed with at least 8 replicate plants for each type of sgRNA. Mock, NB-Cas9OE plants treated with infiltration buffer; Vector Control, TRV2 with a non-viral target sequence;N.A not
applicable.
Treatment / sgRNA Mock experiment
Vector Control experiment
IR-sgRNA / TYLCV
CP-sgRNA / TYLCV
RCRII-sgRNA / TYLCV
IR + CP-sgRNA / TYLCV
IR-CP-sgRNA / TYLCV (PTG)
No. of plants / experiment 8 8 12 8 8 8 10
No. of experimental Repeats 3 3 5 3 3 3 4
Total number of plants 40 40 60 24 24 24 40
No. of Plants with no TYLCV symptoms on leaves
(100 %) (None) (85 %) (None) (11.6 %) (88.6 %) (95 %)
No. of Plants with no TYLCV symptoms on leaves but stunted
(None) (None) (35.8 %) (None) (None) (27.7 %) (7 %)
No. of Plants with Mild TYLCV symptoms
(None) (None) (15.2 %) (73.3 %) (88.7 %) (11.3 %) 3 %
No. of Plants with severe TYLCV symptoms
(None) (100 %) (None) (27.7 %) (12.3 %) (None) (None)
TYLCV detection by PCR (None) (100 %) (39 %) (94 %) (91 %) (25 %) (11 %)
TYLCV detection by DNA blotting
(None) (100 %) (19 %) (68 %) (53 %) (11 %) (3 %)
No. of clones sequenced (None) 30 300 88 142 98(IR) / 58 (CP) 112(IR) / 66(CP)
No. of clones with Indels (None) (None) (36 - 42 %) (22 – 28 %) (31 -39 %) 28 % (IR) and 19 % (CP)
38 % IR and 31 % CP
Can target N.A N.A IR sequence of
TYLCV (38%) BCTV (22%) MeMV (31%)
Specific Specific N.A N.A
